Negative co-operativity in Escherichia coli single strand binding protein-oligonucleotide interactions. II. Salt, temperature and oligonucleotide length effects

Wlodzimierz Bujalowski, Timothy M. Lohman

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Abstract

We have examined the salt and temperature dependences of the equilibrium binding of the Escherichia coli single strand binding (SSB) tetramer to a series of oligodeoxythymidylates, dT(pT)N-1, with N = 16, 28, 35, 56 and 70. Absolute binding isotherms were obtained, based on the quenching of the intrinsic protein fluorescence upon formation of the complexes. The shorter oligonucleotides, with N = 16, 28 and 35, bind to multiple sites on the SSB tetramer and negative co-operativity is observed among these binding sites. We have quantitatively analyzed these isotherms, using a statistical thermodynamic ("square") model to obtain the intrinsic binding constant, KN, and the negative co-operativity constant, σN. For all oligonucleotides, we find that KN decreases significantly with increasing concentration of monovalent salt, indicating a large electrostatic component to the free energy of the interaction (e.g. ∂log K N ∂log [NaBr] = -2.7, -4.6 and -7.1 for N = 16, 35 and 70, respectively), with contributions from both cations and anions. For oligonucleotides that span two or more subunits, there is a significant unfavorable contribution to the binding free energy for each intersubunit crossing, with an accompanying uptake of anions. Therefore, the extent of anion uptake increases as the number of intersubunit crossings increase. There is a strong temperature dependence for the intrinsic binding of dT(pT)15, such that ΔH0 = -26(± 3) kcal/mol dT(pT)15. Negative co-operativity exists under all solution conditions tested, i.e. σN < 1, and this is independent of anion concentration and type. However, the negative co-operativity constant does decrease with decreasing concentration of cation. The dependence of σ16 on Na+ concentration indicates that an average of one sodium ion is taken up as a result of the negative co-operativity between two dT(pT)15 binding sites. These data and the lack of a temperature dependence for σ16 suggest that the molecular basis for the negative co-operativity is predominantly electrostatic and may be due to the repulsion of regions of single-stranded DNA that are required to bind in close proximity on an individual SSB tetramer.

Original languageEnglish (US)
Pages (from-to)269-288
Number of pages20
JournalJournal of Molecular Biology
Volume207
Issue number1
DOIs
StatePublished - May 5 1989
Externally publishedYes

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Oligonucleotides
Anions
Carrier Proteins
Salts
Escherichia coli
Temperature
Static Electricity
Cations
Binding Sites
Single-Stranded DNA
Thermodynamics
Fluorescence
Sodium
Ions
Proteins

ASJC Scopus subject areas

  • Virology

Cite this

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title = "Negative co-operativity in Escherichia coli single strand binding protein-oligonucleotide interactions. II. Salt, temperature and oligonucleotide length effects",
abstract = "We have examined the salt and temperature dependences of the equilibrium binding of the Escherichia coli single strand binding (SSB) tetramer to a series of oligodeoxythymidylates, dT(pT)N-1, with N = 16, 28, 35, 56 and 70. Absolute binding isotherms were obtained, based on the quenching of the intrinsic protein fluorescence upon formation of the complexes. The shorter oligonucleotides, with N = 16, 28 and 35, bind to multiple sites on the SSB tetramer and negative co-operativity is observed among these binding sites. We have quantitatively analyzed these isotherms, using a statistical thermodynamic ({"}square{"}) model to obtain the intrinsic binding constant, KN, and the negative co-operativity constant, σN. For all oligonucleotides, we find that KN decreases significantly with increasing concentration of monovalent salt, indicating a large electrostatic component to the free energy of the interaction (e.g. ∂log K N ∂log [NaBr] = -2.7, -4.6 and -7.1 for N = 16, 35 and 70, respectively), with contributions from both cations and anions. For oligonucleotides that span two or more subunits, there is a significant unfavorable contribution to the binding free energy for each intersubunit crossing, with an accompanying uptake of anions. Therefore, the extent of anion uptake increases as the number of intersubunit crossings increase. There is a strong temperature dependence for the intrinsic binding of dT(pT)15, such that ΔH0 = -26(± 3) kcal/mol dT(pT)15. Negative co-operativity exists under all solution conditions tested, i.e. σN < 1, and this is independent of anion concentration and type. However, the negative co-operativity constant does decrease with decreasing concentration of cation. The dependence of σ16 on Na+ concentration indicates that an average of one sodium ion is taken up as a result of the negative co-operativity between two dT(pT)15 binding sites. These data and the lack of a temperature dependence for σ16 suggest that the molecular basis for the negative co-operativity is predominantly electrostatic and may be due to the repulsion of regions of single-stranded DNA that are required to bind in close proximity on an individual SSB tetramer.",
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T1 - Negative co-operativity in Escherichia coli single strand binding protein-oligonucleotide interactions. II. Salt, temperature and oligonucleotide length effects

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N2 - We have examined the salt and temperature dependences of the equilibrium binding of the Escherichia coli single strand binding (SSB) tetramer to a series of oligodeoxythymidylates, dT(pT)N-1, with N = 16, 28, 35, 56 and 70. Absolute binding isotherms were obtained, based on the quenching of the intrinsic protein fluorescence upon formation of the complexes. The shorter oligonucleotides, with N = 16, 28 and 35, bind to multiple sites on the SSB tetramer and negative co-operativity is observed among these binding sites. We have quantitatively analyzed these isotherms, using a statistical thermodynamic ("square") model to obtain the intrinsic binding constant, KN, and the negative co-operativity constant, σN. For all oligonucleotides, we find that KN decreases significantly with increasing concentration of monovalent salt, indicating a large electrostatic component to the free energy of the interaction (e.g. ∂log K N ∂log [NaBr] = -2.7, -4.6 and -7.1 for N = 16, 35 and 70, respectively), with contributions from both cations and anions. For oligonucleotides that span two or more subunits, there is a significant unfavorable contribution to the binding free energy for each intersubunit crossing, with an accompanying uptake of anions. Therefore, the extent of anion uptake increases as the number of intersubunit crossings increase. There is a strong temperature dependence for the intrinsic binding of dT(pT)15, such that ΔH0 = -26(± 3) kcal/mol dT(pT)15. Negative co-operativity exists under all solution conditions tested, i.e. σN < 1, and this is independent of anion concentration and type. However, the negative co-operativity constant does decrease with decreasing concentration of cation. The dependence of σ16 on Na+ concentration indicates that an average of one sodium ion is taken up as a result of the negative co-operativity between two dT(pT)15 binding sites. These data and the lack of a temperature dependence for σ16 suggest that the molecular basis for the negative co-operativity is predominantly electrostatic and may be due to the repulsion of regions of single-stranded DNA that are required to bind in close proximity on an individual SSB tetramer.

AB - We have examined the salt and temperature dependences of the equilibrium binding of the Escherichia coli single strand binding (SSB) tetramer to a series of oligodeoxythymidylates, dT(pT)N-1, with N = 16, 28, 35, 56 and 70. Absolute binding isotherms were obtained, based on the quenching of the intrinsic protein fluorescence upon formation of the complexes. The shorter oligonucleotides, with N = 16, 28 and 35, bind to multiple sites on the SSB tetramer and negative co-operativity is observed among these binding sites. We have quantitatively analyzed these isotherms, using a statistical thermodynamic ("square") model to obtain the intrinsic binding constant, KN, and the negative co-operativity constant, σN. For all oligonucleotides, we find that KN decreases significantly with increasing concentration of monovalent salt, indicating a large electrostatic component to the free energy of the interaction (e.g. ∂log K N ∂log [NaBr] = -2.7, -4.6 and -7.1 for N = 16, 35 and 70, respectively), with contributions from both cations and anions. For oligonucleotides that span two or more subunits, there is a significant unfavorable contribution to the binding free energy for each intersubunit crossing, with an accompanying uptake of anions. Therefore, the extent of anion uptake increases as the number of intersubunit crossings increase. There is a strong temperature dependence for the intrinsic binding of dT(pT)15, such that ΔH0 = -26(± 3) kcal/mol dT(pT)15. Negative co-operativity exists under all solution conditions tested, i.e. σN < 1, and this is independent of anion concentration and type. However, the negative co-operativity constant does decrease with decreasing concentration of cation. The dependence of σ16 on Na+ concentration indicates that an average of one sodium ion is taken up as a result of the negative co-operativity between two dT(pT)15 binding sites. These data and the lack of a temperature dependence for σ16 suggest that the molecular basis for the negative co-operativity is predominantly electrostatic and may be due to the repulsion of regions of single-stranded DNA that are required to bind in close proximity on an individual SSB tetramer.

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